Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate

Abstract

Isolated intact lamb liver cells were prepared from 24 h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase [EC 3.4.24.3]. Isolated caudate-lobe liver cells showed rates of gluconeogenesis from 10 mM-propionate and 10 mM-lactate similar to rates determined in isolated median-lobe cells and with rates determined with the isolated perfused lamb liver. The gluconeogenic potential of substrates tested was age dependent. Cells prepared from suckling lambs (up to 20 days of age and essentially non-ruminant) showed highest rates from galactose, serine and alanine; those prepared from post-weaned lambs (older than 30 days of age and ruminant) showed highest rates from propionate, lactate and fructose. Gluconeogenic rates from endogenous precursors, 10 mM-propionate and 10 mM-galactose, were linear for 1 h and were both stimulated by 1 .mu.M-glucagon. Provided the endogenous rate of gluconeogenesis remained unchanged after substrate addition, glucagon caused a net stimulation of gluconeogensis from each of these substrates. Gluconeogenic capacity and glucagon sensitivity were examined in cells maintained in substrate-free oxygenated buffer at 37.degree., 22.degree. and 0.degree. C. Under optimum conditions of storage (i.e., at 22.degree. C in gelatin-containing buffer) deterioration of the lamb cells still proceeded rapidly. Loss of glucagon responsiveness preceded the loss of ability to convert precursor into glucose. n-Butyric acid, 2-methylpropanoic acid and 3-methylbutanoic acid at concentrations similar to those found in portal-vein blood stimulated gluconeogensis from 10 mM-galactose or 10 mM-propionate. Gluconeogenesis from galactose was stimulated to the greater extent. Glucagon (1 .mu.M) and 2mM-butyrate accelerated the rate of glucose formation in liver cells of 24 h-starved animals from lactate + pyruvate or fructose. Insulin (20 nM) decreased both gluconeogenesis and the efficacy of 1 .mu.M-glucagon. For lactate + pyruvate as substrate, the stimulatory effect of butyrate was additive to that of 1 .mu.M-glucagon, and for both lactate + pyruvate and fructose the stimulatory effect of butyrate was not influenced by 20 nM-insulin. In contrast with glucagon, which stimulated the rate of glycogenolysis in cells prepared from fed lambs, butyrate (0.1-20 mM) had no effect. Glucagon and butyrate apparently stimulate lamb live-cell gluconeogenesis by different mechanisms.